Synthetic olgononucleotides for detection of nucleic acid binding proteins

ABSTRACT

Synthetic oligonucleotides that comprise a nucleic acid binding protein binding site, PCR primer sequences, and tag sequences that do not bind to nucleic acid binding proteins, with a total length of 85-130 nucleotides are disclosed herein. Also disclosed are libraries and kits comprising the synthetic oligonucleotides as well as methods of detecting nucleic acid binding proteins in a sample using the synthetic oligonucleotides.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Provisional PatentApplication No. 61/870,904, entitled “SYNTHETIC OLIGONUCLEOTIDES FORDETECTION OF NUCLEIC ACID BINDING PROTEINS,” filed Aug. 28, 2013, thedisclosure of which is incorporated herein by reference in its entirety.

FIELD

Generally, the field is nucleic acid probes and arrays thereof. Morespecifically, the field is nucleic acid probes that can detect,identify, and quantify nucleic acid binding proteins in a sample.

BACKGROUND

Nucleic acid binding proteins are proteins in the nucleus that bind tonucleic acids such as DNA and RNA to perform any of a number ofactivities, including the promotion or inhibition of RNA transcriptionor protein translation. Their binding can be identified with DNA probesthat are separated and rendered visible by gel electrophoresis but theirdetection is limited to one or several binding proteins at one time.Using current methods, it is difficult to quantify nucleic acid bindingproteins present in a biological sample. Many currently availabletechnologies comprise the use of probes that self-hybridize, thereforereducing specificity and ease of use. Clearly, new probes that identifynucleic acid binding proteins in biological samples are necessary.

SUMMARY

Disclosed herein are synthetic oligonucleotides that identify DNAbinding proteins in a sample. This includes methods of using saidoligonucleotides to measure the binding action of a plurality of DNAbinding proteins by binding the oligonucleotides to a microarray chip.

The synthetic oligonucleotide comprises sequences that include a nucleicacid binding protein binding site, a sequence that is an RNA polymerasepromoter or a binding site for a PCR primer, and a tag sequence thatdoes not bind any nucleic acid binding protein. At least the nucleicacid binding protein binding site is double stranded and the entiresynthetic oligonucleotide can be double stranded. The probe is between85 and 130 base pairs in length, including between 90 and 98 nucleotidesin length. The sequence may comprise T7, SP6, or T5 promoters includingSEQ ID NO: 1 and SEQ ID NO: 2. In some examples, the tag sequence is anyof SEQ ID NOs: 3-98.

Further disclosed are sets of synthetic oligonucleotides each withdifferent nucleic acid binding protein binding sites as well as kitscomprising sets of synthetic oligonucleotides and microarrays comprisingoligonucleotides complementary to the tag sequences. Theoligonucleotides on the microarray are addressable.

Also disclosed is a method of detecting a DNA binding protein in asample involving contacting one of the disclosed syntheticoligonucleotides with a sample. The sample is then subjected toconditions that allow binding of any DNA binding proteins in the sampleto the synthetic oligonucleotides to form protein/oligonucleotidecomplexes. The protein/oligonucleotide complexes are purified byelectrophoresis. The oligonucleotides are labeled and hybridized to anarray. Detection of the label within the context of the array indicatesthe presence of the DNA binding protein in the sample. The label may beany label, including a fluorescent label. The label may be incorporatedinto the oligonucleotide by any method including method involving use ofRNA polymerase or polymerase chain reaction. Electrophoresis may beperformed using any device or method including the devices and methodsdescribed in US2012/0160683 which is hereby incorporated by reference inits entirety.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying FIGUREand sequence listing.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a bar graph of the relative strength (Y axis) of threedifferent probes (Probe 1—SEQ ID NO: 197) (Probe 2—SEQ ID NO: 198) andProbe 3—SEQ ID NO: 99 incubated with nuclear extracts comprising theindicated amounts of added nuclear factor kappa-light-chain enhance ofactivated B cells (NF-κB), purified by electrophoresis, and bound to amicroarray as described herein.

BRIEF DESCRIPTION OF SEQUENCES

The nucleic acid sequences listed in the accompanying sequence listingare shown using standard letter abbreviations for nucleotide bases, andthree letter code for amino acids, as defined in 37 C.F.R. 1.822. Onlyone strand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by any reference to the displayedstrand. All sequence database accession numbers referenced herein areunderstood to refer to the version of the sequence identified by thataccession number as it was available on the designated date. In theaccompanying sequence listing:

SEQ ID NO: 1 is a universal forward primer sequence binding site.

SEQ ID NO: 2 is a universal reverse primer sequence binding site.

SEQ ID NOs: 3-98 are examples of tag sequences

SEQ ID NOs: 99-100 are synthetic oligonucleotide probes that can bindNF-κB

SEQ ID NOs: 101-102 are synthetic oligonucleotide probes that can bindP53.

SEQ ID NOs: 103-104 are synthetic oligonucleotide probes that can bindAR (androgen receptor).

SEQ ID NOs: 105-106 are synthetic oligonucleotide probes that can bindCreb.

SEQ ID NOs: 107-108 are synthetic oligonucleotide probes that can bindactivator protein 1 (AP-1).

SEQ ID NOs: 109-110 are synthetic oligonucleotide probes that can bindearly growth response protein 1(EGR-1).

SEQ ID NOs: 111-112 are synthetic oligonucleotide probes that can bindactivating protein 2 (AP-2).

SEQ ID NOs: 113-114 are synthetic oligonucleotide probes that can bindNkx-3.1.

SEQ ID NOs: 115-116 are synthetic oligonucleotide probes that can bindprostate-specific antigen (PSA).

SEQ ID NOs: 117-118 are synthetic oligonucleotide probes that can bindC-myb.

SEQ ID NOs: 119-120 are synthetic oligonucleotide probes that can bindperoxisome proliferator-activated receptor (PPAR).

SEQ ID NOs: 121-122 are synthetic oligonucleotide probes that can bindPPAR gamma.

SEQ ID NOs: 123-124 are synthetic oligonucleotide probes that can bindOct-1.

SEQ ID NOs: 125-126 are synthetic oligonucleotide probes that can bindhypoxia-inducible factor 1 alpha (HIF-1α).

SEQ ID NOs: 127-128 are synthetic oligonucleotide probes that can bindE2F transcription factor 1 (E2F-1).

SEQ ID NOs: 129-130 are synthetic oligonucleotide probes that can bindCEBP (CCAAT-enhancer binding protein).

SEQ ID NOs: 131-132 are synthetic oligonucleotide probes that can bindB-cell lymphoma 6 (Bcl-6).

SEQ ID NOs: 133-134 are synthetic oligonucleotide probes that can bindSRE (sterol regulatory element).

SEQ ID NOs: 135-136 are synthetic oligonucleotide probes that can bindOxo3a.

SEQ ID NOs: 137-138 are synthetic oligonucleotide probes that can bindforkhead box a (Foxa).

SEQ ID NO: 139 and 140 are synthetic oligonucleotide probes that canbind forkhead box o (Foxo).

SEQ ID NOs: 141 and 142 are synthetic oligonucleotide probes that canbind PR (progesterone receptor).

SEQ ID NOs: 143 and 144 are synthetic oligonucleotide probes that canbind RAR (retinoic acid receptor).

SEQ ID NOs: 145 and 146 are synthetic oligonucleotide probes that canbind Snai1.

SEQ ID NOs: 147 and 148 are synthetic oligonucleotide probes that canbind Stat1.

SEQ ID NOs: 149 and 150 are synthetic oligonucleotide probes that canbind Stat3.

SEQ ID NOs: 151 and 152 are synthetic oligonucleotide probes that canbind Stat4.

SEQ ID NOs: 153 and 154 are synthetic oligonucleotide probes that canbind Stat5.

SEQ ID NOs: 155 and 156 are synthetic oligonucleotide probes that canbind Stat5 and Stat6.

SEQ ID NOs: 157 and 158 are synthetic oligonucleotide probes that canbind Brn-3.

SEQ ID NOs: 159 and 160 are synthetic oligonucleotide probes that canbind CBF (CCAAT binding factor).

SEQ ID NOs: 161 and 162 are synthetic oligonucleotide probes that canbind CDP (CCAAT displacement protein).

SEQ ID NOs: 163 and 164 are synthetic oligonucleotide probes that canbind CCCTC-binding factor (CTCF).

SEQ ID NO: 165 and 166 are synthetic oligonucleotide probes that canbind Fast-1.

SEQ ID NOs: 167 and 168 are synthetic oligonucleotide probes that canbind GATA binding protein 2 (GATA2).

SEQ ID NOs: 169 and 170 are synthetic oligonucleotide probes that canbind runt-related transcription factor 3 (RUNX3).

SEQ ID NOs: 171 and 172 are synthetic oligonucleotide probes that canbind ETS-related gene (ERG).

SEQ ID NOs: 173 and 174 are synthetic oligonucleotide probes that canbind FLI-1 (Friend leukemia virus integration 1).

SEQ ID NOs: 175 and 176 are synthetic oligonucleotide probes that canbind hepatocyte nuclear factor 4 (HNF-4).

SEQ ID NOs: 177 and 178 are synthetic oligonucleotide probes that canbind IRF-1 (interferon regulatory factor 1).

SEQ ID NOs: 179 and 180 is a synthetic oligonucleotide probe that canbind nuclear factor 1 (NF-1).

SEQ ID NOs: 181 and 182 are synthetic oligonucleotide probes that canbind nuclear factor, erythroid 2 (NF-e2).

SEQ ID NOs: 183 and 184 are synthetic oligonucleotide probes that canbind upstream stimulatory factor (USF-1).

SEQ ID NO: 185 is a synthetic oligonucleotide probe that can bindInterferon-Gamma Activated Sequence (GAS)/Interferon-Stimulated ResponseElement (ISRE).

SEQ ID NO: 186 is a synthetic oligonucleotide probe that can bind Smad.

SEQ ID NOs: 187-188 are synthetic oligonucleotide probes that can bindSmad.

SEQ ID NO: 189-192 are synthetic oligonucleotide probes that lack anucleic acid binding protein binding site used as negative controls.

SEQ ID NOs: 193 and 194 are synthetic oligonucleotide probes that canbind Myc-Max.

SEQ ID NO: 195 is a T7 promoter site

SEQ ID NO: 196 is an SP6 promoter site.

SEQ ID NOs: 197-199 are synthetic oligonucleotides that can bind NF-Kb.

SEQ ID NOs: 200-202 are synthetic oligonucleotides with mutated NF-Kbbinding sites.

SEQ ID NOs: 203-205 are synthetic oligonucleotides that can bindestrogen receptor.

SEQ ID NOs: 206-208 are synthetic oligonucleotides with mutant estrogenreceptor binding sites.

SEQ ID NOs: 209-211 are synthetic oligonucleotides that can bind SP1

SEQ ID NOs: 212-214 are synthetic oligonucleotides with mutant SP1binding sites.

DETAILED DESCRIPTION I. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

To facilitate review of the various embodiments of this disclosure, thefollowing explanations of specific terms are provided:

Amplifying a Nucleic Acid Molecule:

to increase the number of copies of a nucleic acid molecule, such as adouble stranded synthetic oligonucleotide described herein. Theresulting products are called amplification products. An example of invitro amplification is the polymerase chain reaction (PCR). Otherexamples of in vitro amplification techniques include quantitativereal-time PCR, strand displacement amplification (see U.S. Pat. No.5,744,311); transcription-free isothermal amplification (see U.S. Pat.No. 6,033,881); repair chain reaction amplification (see WO90/01069);ligase chain reaction amplification (see EP application 320 308); gapfilling ligase chain reaction amplification (see U.S. Pat. No.5,427,930); coupled ligase detection and PCR (see U.S. Pat. No.6,027,889); and NASBA™ RNA transcription-free amplification (see U.S.Pat. No. 6,025,134).

Amplification of a nucleic acid molecule may also include the productionof RNA molecules from a DNA template through a transcription reaction(such as an in vitro transcription reaction). In this case, a sequenceto be amplified comprises an RNA polymerase promoter that binds to anRNA polymerase which, in the presence of ribonucleotides, producesmultiple copies of an RNA sequence.

Amplification of a nucleic acid sequence may be used for any of a numberof purposes, including increasing the amount of a rare sequence to beanalyzed by other methods. It may also be used to identify a sequencedirectly (for example, though an amplification refractory mutationsystem) or as part of a DNA sequencing method.

Array:

An arrangement of molecules, such as biological macromolecules (such aspeptides, antibodies, or nucleic acid molecules) or biological samples(such as tissue sections), in addressable locations on or in a solidsupport or substrate. A “microarray” is an array that is miniaturized soas to require or be aided by microscopic examination for evaluation oranalysis. Arrays are sometimes called chips or biochips. The array ofmolecules (“features”) makes it possible to carry out a large number ofanalyses on a sample at one time. In certain example arrays, one or moremolecules (such as an oligonucleotide probe) will occur on the array aplurality of times (such as two or three times), for instance to provideinternal controls. The number of addressable locations on the array canvary, for example from at least one, to at least 2, to at least 5, to atleast 10, at least 20, at least 30, at least 50, at least 75, at least100, at least 150, at least 200, at least 300, at least 500, least 550,at least 600, at least 800, at least 1000, at least 10,000, at least100,000, or more. In particular examples, an array includes nucleic acidmolecules, such as oligonucleotide sequences that are at least 10nucleotides in length.

In particular examples, the array may comprise an oligonucleotide thatcan hybridize to a tag sequence on a nucleic acid probe. In furtherexamples, the array may be a universal array that can bind a set oftags. Such an array might be stripped and reused as appropriate (see USPatent Application Publication Number 2009/0061424 which is incorporatedby reference herein.)

Within an array, each arrayed biomolecule is addressable, in that itslocation can be reliably and consistently determined within at least twodimensions of the array. The feature application location on an arraycan assume different shapes. For example, the array can be regular (suchas arranged in uniform rows and columns) or irregular. Thus, in orderedarrays the location of each sample is assigned to the sample at the timewhen it is applied to the array, and a key may be provided in order tocorrelate each location with the appropriate target or feature position.Often, ordered arrays are arranged in a symmetrical grid pattern, butsamples could be arranged in other patterns (such as in radiallydistributed lines, spiral lines, or ordered clusters). Addressablearrays usually are computer readable, in that a computer can beprogrammed to correlate a particular address on the array withinformation about the sample at that position (such as hybridization orbinding data, including for instance signal intensity). In some examplesof computer readable formats, the individual features in the array arearranged regularly, for instance in a Cartesian grid pattern, which canbe correlated to address information by a computer.

Binding or Stable Binding:

Physical methods of detecting the binding of complementary strands ofnucleic acid molecules, include but are not limited to, such methods asDNase I or chemical footprinting, gel shift and affinity cleavageassays, Northern blotting, dot blotting and light absorption detectionprocedures. For example, one method involves observing a change in lightabsorption of a solution containing an oligonucleotide (or an analog)and a target nucleic acid at 220 to 300 nm as the temperature is slowlyincreased. If the oligonucleotide or analog has bound to its target,there is an increase in absorption at a characteristic temperature asthe oligonucleotide (or analog) and target disassociate from each other,or melt. In another example, the method involves detecting a signal,such as a detectable label, present on one or both nucleic acidmolecules. The binding between an oligomer and its target nucleic acidis frequently characterized by the temperature (T_(m)) at which 50% ofthe oligomer is melted from its target. A higher T_(m) indicates astronger or more stable complex relative to a complex with a lowerT_(m).

Buffer Solution:

An aqueous solution consisting of a mixture of a weak acid and itsconjugate base or a weak base and its conjugate acid. It has theproperty that the pH of the solution changes very little when a smallamount of acid or base is added to it. Buffer solutions can keep pH at anearly constant value in a wide variety of chemical applications.

Contacting:

Placement in direct physical association; includes solid, liquid, andgaseous associations. Contacting includes contact between one moleculeand another molecule. Contacting can occur in vitro with isolated cellsor tissue or in vivo by administering to a subject. The concept ofcontacting may also be encompassed by adding a molecule to a solid,liquid, or gaseous mixture.

Control:

A control may be any sample or standard used for comparison with anexperimental sample. In some examples, the control is a sample obtainedfrom a healthy patient or a non-tumor tissue sample obtained from apatient diagnosed with cancer (such as non-tumor tissue adjacent to thetumor). In some examples, the control is a historical control orstandard reference value or range of values (such as a previously testedcontrol sample, such as a group of cancer patients with poor prognosis,or group of samples that represent baseline or normal values, such asthe level of one or more of the genes disclosed herein in non-tumortissue). A control may also serve as a threshold level of expression ofa biomarker that indicates a particular disease outcome.

Electrophoresis:

The process of separating a mixture of charged molecules based on thedifferent mobility of these charged molecules in response to an appliedelectric current. A particular type of electrophoresis is gelelectrophoresis. The mobility of a molecule is generally related to thecharacteristics of the charged molecule, such as size, shape, andsurface charge amongst others. The mobility of a molecule also isinfluenced by the electrophoretic medium, for example the composition ofthe electrophoresis gel. For example, when the electrophoretic medium iscross-linked acrylamide (polyacrylamide) increasing the percentage ifacrylamide in the gel reduces the size of the resulting pores in the geland retards the mobility of a molecule relative to a gel with a lowerpercentage of acrylamide (larger pore size). Gel electrophoresis can beperformed for analytical purposes, but can be used as a preparativetechnique to partially purify molecules prior to use of other methods,such as mass spectrometry, PCR, cloning, DNA sequencing, array analysis,and immuno-blotting.

Label:

A detectable compound or composition that is conjugated directly orindirectly to another molecule (such as an oligonucleotide) tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent molecules, enzymatic linkages andradioactive isotopes.

Nucleic Acid Molecules:

A deoxyribonucleotide or ribonucleotide polymer including, withoutlimitation, cDNA, mRNA, genomic DNA, methylated DNA, and synthetic (suchas chemically synthesized) nucleic acids such as DNA, RNA, and/ormethylated oligonucleotides. The nucleic acid molecule can bedouble-stranded or single-stranded. Where single-stranded, the nucleicacid molecule can be the sense strand or the antisense strand. Inaddition, nucleic acid molecule can be circular or linear. A nucleicacid molecule may also be termed a polynucleotide and the terms are usedinterchangeably.

Oligonucleotide:

A plurality of joined nucleotides joined by native phosphodiester bonds,between about 6 and about 300 nucleotides in length. An oligonucleotideanalog refers to moieties that function similarly to oligonucleotidesbut have non-naturally occurring portions. For example, oligonucleotideanalogs can contain non-naturally occurring portions, such as alteredsugar moieties or inter-sugar linkages, such as a phosphorothioateoligodeoxynucleotide.

Particular oligonucleotides and oligonucleotide analogs can includelinear sequences up to about 200 nucleotides in length, for example asequence (such as DNA or RNA) that is at least 6 nucleotides, forexample at least 8, at least 10, at least 15, at least 20, at least 21,at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 100 or even at least 200 nucleotides long, including85-130 nucleotides long.

An oligonucleotide can be used to detect the presence of a complementarysequence by molecular hybridization. Such an oligonucleotide can also betermed a probe. In particular examples, such oligonucleotides include alabel that permits detection of oligonucleotide probe:target sequencehybridization complexes. In a particular example, a probe includes atleast one fluorophore, such as an acceptor fluorophore or donorfluorophore. For example, a fluorophore can be attached at the 5′- or3′-end of the probe. In specific examples, the fluorophore is attachedto the base at the 5′-end of the probe, the base at its 3′-end, thephosphate group at its 5′-end or a modified base, such as a T internalto the probe.

An oligonucleotide can be used to prime a nucleic acid amplification.Such an oligonucleotide may also be termed a primer. An oligonucleotideprimer can be annealed to a complementary target nucleic acid moleculeby nucleic acid hybridization to form a hybrid between the primer andthe target nucleic acid strand. A primer can be extended along thetarget nucleic acid molecule by a polymerase enzyme.

The specificity of an oligonucleotide primer increases with its length.Thus, for example, a primer that includes 30 consecutive nucleotideswill anneal to a target sequence with a higher specificity than acorresponding primer of only 15 nucleotides. Thus, to obtain greaterspecificity, probes and primers can be selected that include at least15, 20, 25, 30, 35, 40, 45, 50, 85, 100, or 120 or more consecutivenucleotides. In particular examples, a primer is at least 15 nucleotidesin length, such as at least 15 contiguous nucleotides complementary to atarget nucleic acid molecule.

Primer pairs can be used for amplification of a nucleic acid sequence,for example, by PCR, real-time PCR, or other nucleic-acid amplificationmethods known in the art. An “upstream” or “forward” primer is a primer5′ to a reference point on a nucleic acid sequence. A “downstream” or“reverse” primer is a primer 3′ to a reference point on a nucleic acidsequence. In general, at least one forward and one reverse primer areincluded in an amplification reaction.

Nucleic acid probes and/or primers can be readily prepared based on thenucleic acid molecules provided herein. PCR primer pairs and probes canbe derived from a known sequence for example, by using any of a numberof computer programs intended for that purpose such as Primer (Version0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge,Mass.) or PRIMER EXPRESS® Software (Applied Biosystems, Foster City,Calif.).

Methods for preparing and using oligonucleotide and other nucleic acidprobes and primers and methods for labeling and guidance in the choiceof labels appropriate for various purposes are described, for example,in Sambrook et al (In Molecular Cloning: A Laboratory Manual, CSHL, NewYork, 1989), Ausubel et al (ed.) (In Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1998), and Innis et al (PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.,San Diego, Calif., 1990).

Promoter:

An array of nucleic acid control sequences, which directs transcriptionof a nucleic acid. Typically, a eukaryotic a promoter includes necessarynucleic acid sequences near the start site of transcription, such as, inthe case of a polymerase II type promoter, a TATA element. A promoteralso optionally includes distal enhancer or repressor elements, whichcan be located as much as several thousand base pairs from the startsite of transcription, such as specific DNA sequences that arerecognized by proteins known as transcription factors.

In prokaryotes, a promoter is recognized by RNA polymerase and anassociated sigma factor, which in turn are brought to the promoter DNAby an activator protein binding to its own DNA sequence nearby.

Sample (or Biological Sample):

A specimen containing genomic DNA, RNA (including mRNA), protein, orcombinations thereof, obtained from a subject. As used herein,biological samples include cells, tissues, and bodily fluids, such as:blood; derivatives and fractions of blood, such as plasma or serum;extracted galls; biopsied or surgically removed tissue, includingtissues that are, for example, unfixed, frozen, fixed in formalin and/orembedded in paraffin; tears; milk; skin scrapes; surface washings;urine; sputum; cerebrospinal fluid; prostate fluid; pus; or bone marrowaspirates. In a particular example, a sample includes a tumor biopsy(such as a prostate tumor biopsy). In another example, a sample includescirculating tumor cells, such as tumor cells present in blood of asubject with a tumor.

Obtaining a biological sample includes, but need not be limited to anymethod of collecting a particular sample known in the art. Obtaining abiological sample from a subject also encompasses receiving a samplethat was collected at a different location than where a method isperformed; receiving a sample that was collected by a differentindividual than an individual that performs the method, receiving asample that was collected at any time period prior to the performance ofthe method, receiving a sample that was collected using a differentinstrument than the instrument that performs the method, or anycombination of these. Obtaining a biological sample from a subject alsoencompasses situations in which the collection of the sample andperformance of the method are performed at the same location, by thesame individual, at the same time, using the same instrument, or anycombination of these.

A biological sample encompasses any fraction of a biological sample orany component of a biological sample that may be isolated and/orpurified from the biological sample. For example: when cells areisolated from blood or tissue, including specific cell types sorted onthe basis of biomarker expression; or when nucleic acid or protein ispurified from a fluid or tissue; or when blood is separated intofractions such as plasma, serum, buffy coat PBMC's or other cellular andnon-cellular fractions on the basis of centrifugation and/or filtration.A biological sample further encompasses biological samples or fractionsor components thereof that have undergone a transformation of mater orany other manipulation. For example, a cDNA molecule made from reversetranscription of mRNA purified from a biological sample may be termed abiological sample.

A biological sample may be obtained from any source including anyorganism (living, dead, or extinct) or any cells derived from anorganism or any artificial cells that may comprise nucleic acids and/ornucleic acid binding proteins. Examples include animals, plants,bacteria, archea, fungi, prions, viruses, or any other source.Biological samples may also include strata that may contain nucleicacids or nucleic acid binding proteins including water, soil, air,fossilized material, including strata from both terrestrial andextraterrestrial origin.

Transcription Factor:

A protein that regulates transcription. In particular, transcriptionfactors regulate the binding of RNA polymerase and the initiation oftranscription. A transcription factor binds upstream or downstream toeither enhance or repress transcription of a gene by assisting orblocking RNA polymerase binding. The term transcription factor includesboth inactive and activated transcription factors.

Transcription factors are typically modular proteins that affectregulation of gene expression. Exemplary transcription factors includeAAF, ab1, ADA2, ADA-NF1, AF-1, AFP1, AhR, AIIN3, ALL-1, alpha-CBF,alpha-CP1, alpha-CP2a, alpha-CP2b, alphaHo, alphaH2-alphaH3, Alx-4,aMEF-2, AML1, AML1a, AML1b, AML1c, AML1DeltaN, AML2, AML3, AML3a, AML3b,AMY-1L, A-Myb, ANF, AP-1, AP-2alphaA, AP-2alphaB, AP-2beta, AP-2gamma,AP-3 (1), AP-3 (2), AP-4, AP-5, APC, AR, AREB6, Arnt, Arnt (774 M form),ARP-1, ATBF1-A, ATBF1-B, ATF, ATF-1, ATF-2, ATF-3, ATF-3deltaZIP, ATF-a,ATF-adelta, ATPF1, Barh11, Barh12, Barx1, Barx2, Bc1-3, BCL-6, BD73,beta-catenin, Bin1, B-Myb, BP1, BP2, brahma, BRCA1, Brn-3a, Brn-3b,Brn-4, BTEB, BTEB2, B-TFIID, C/EBPalpha, C/EBPbeta, C/EBPdelta,CACCbinding factor, Cart-1, CBF (4), CBF (5), CBP, CCAAT-binding factor,CCMT-binding factor, CCF, CCG1, CCK-1a, CCK-1b, CD28RC, cdk2, cdk9,Cdx-1, CDX2, Cdx-4, CFF, Chx1O, CLIM1, CLIM2, CNBP, CoS, COUP, CP1,CP1A, CP1C, CP2, CPBP, CPE binding protein, CREB, CREB-2, CRE-BP1,CRE-BPa, CREMalpha, CRF, Crx, CSBP-1, CTCF, CTF, CTF-1, CTF-2, CTF-3,CTF-5, CTF-7, CUP, CUTL1, Cx, cyclin A, cyclin T1, cyclin T2, cyclinT2a, cyclin T2b, DAP, DAX1, DB1, DBF4, DBP, DbpA, DbpAv, DbpB, DDB,DDB-1, DDB-2, DEF, deltaCREB, deltaMax, DF-1, DF-2, DF-3, Dlx-1, Dlx-2,Dlx-3, DIx4 (long isoform), Dlx-4 (short isoform, Dlx-5, Dlx-6, DP-1,DP-2, DSIF, DSIF-β14, DSIF-β160, DTF, DUX1, DUX2, DUX3, DUX4, E, E12,E2F, E2F+E4, E2F+p107, E2F-1, E2F-2, E2F-3, E2F-4, E2F-5, E2F-6, E47,E4BP4, E4F, E4F1, E4TF2, EAR2, EBP-80, EC2, EF1, EF-C, EGR1, EGR2, EGR3,EIIaE-A, EIIaE-B, EIIaE-Calpha, EIIaE-Cbeta, EivF, EIf-1, Elk-1, Emx-1,Emx-2, Emx-2, En-1, En-2, ENH-bind. prot., ENKTF-1, EPAS1, epsilonF1,ER, Erg-1, Erg-2, ERR1, ERR2, ETF, Ets-1, Ets-1 deltaVi1, Ets-2, Evx-1,F2F, factor 2, Factor name, FBP, f-EBP, FKBP59, FKHL18, FKHRL1P2, Fli-1,Fos, FOXB1, FOXC1, FOXC2, FOXD1, FOXD2, FOXD3, FOXD4, FOXE1, FOXE3,FOXF1, FOXF2, FOXG1a, FOXG1b, FOXG1c, FOXH1, FOXI1, FOXJ1a, FOXJ1b,FOXJ2 (long isoform), FOXJ2 (short isoform), FOXJ3, FOXK1a, FOXK1b,FOXK1c, FOXL1, FOXM1a, FOXM1b, FOXM1c, FOXN1, FOXN2, FOXN3, FOX01a,FOX01b, FOXO2, FOXO3a, FOXO3b, FOXO4, FOXP1, FOXP3, Fra-1, Fra-2, FTF,FTS, G factor, G6 factor, GABP, GABP-alpha, GABP-beta1, GABP-beta2, GADD153, GAF, gammaCMT, gammaCAC1, gammaCAC2, GATA-1, GATA-2, GATA-3,GATA-4, GATA-5, GATA-6, Gbx-1, Gbx-2, GCF, GCMa, GCNS, GF1, GLI, GLI3,GR alpha, GR beta, GRF-1, Gsc, Gsc1, GT-IC, GT-IIA, GT-IIBalpha,GT-IlBbeta, H1TF1, H1TF2, H2RIIBP, H4TF-1, H4TF-2, HAND1, HAND2, HB9,HDAC1, HDAC2, HDAC3, hDaxx, heat-induced factor, HEB, HEB1-p67,HEB1-p94, HEF-1 B, HEF-1T, HEF-4C, HEN1, HEN2, Hesx1, Hex, HIF-1,HIF-1alpha, HIF-1beta, HiNF-A, HiNF-B, HINF-C, HINF-D, HiNF-D3, HiNF-E,HiNF-P, HIP1, HIV-EP2, Hlf, HLTF, HLTF (Met123), HLX, HMBP, HMG I, HMGI(Y), HMG Y, HMGI-C, HNF-1A, HNF-1B, HNF-1C, HNF-3, HNF-3alpha,HNF-3beta, HNF-3gamma, HNF4, HNF-4-alpha, HNF4alpha1, HNF-4-alpha2,HNF-4-alpha3, HNF-4-alpha4, HNF4gamma, HNF-6alpha, hnRNP K, HOX11,HOXA1, HOXA10, HOXA10 PL2, HOXA11, HOXA13, HOXA2, HOXA3, HOXA4, HOXA5,HOXA6, HOXA7, HOXA9A, HOXA9B, HOXB-1, HOXB13, HOXB2, HOXB3, HOXB4,HOXB5, HOXB6, HOXA5, HOXB7, HOXB8, HOXB9, HOXC10, HOXC11, HOXC12,HOXC13, HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXD10, HOXD11, HOXD12,HOXD13, HOXD3, HOXD4, HOXD8, HOXD9, Hp55, Hp65, HPX42B, HrpF, HSF, HSF1(long), HSF1 (short), HSF2, hsp56, Hsp90, IBP-1, ICER-II, ICER-ligamma,ICSBP, Id1, Id1 H′, Id2, Id3, Id3/Heir-1, IF1, IgPE-1, IgPE-2, IgPE-3,IkappaB, IkappaB-alpha, IkappaB-beta, IkappaBR, II-1 RF, IL-6 RE-BP,11-6 RF, INSAF, IPF1, IRF-1, IRF-2, ir1B, IRX2a, Irx-3, lrx-4, ISGF-1,ISGF-3, ISGF3alpha, ISGF-3gamma, lsl-1, ITF, ITF-1, ITF-2, JRF, Jun,JunB, JunD, kappay factor, KBP-1, KER1, KER-1, Kox1, KRF-1, Kuautoantigen, KUP, LBP-1, LBP-1a, LBX1, LCR-F1, LEF-1, LEF-1B, LF-A1,LHX1, LHX2, LHX3a, LHX3b, LHX5, LHX6.1a, LHX6.1b, LIT-1, Lmo1, Lmo2,LMX1A, LMX1B, L-My1 (long form), L-My1 (short form), L-My2, LSF,LXRalpha, LyF-1, LyI-1, M factor, Mad1, MASH-1, Max1, Max2, MAZ, MAZ1,MB67, MBF1, MBF2, MBF3, MBP-1 (1), MBP-1 (2), MBP-2, MDBP, MEF-2,MEF-2B, MEF-2C (433 AA form), MEF-2C (465 AA form), MEF-2C (473 M form),MEF-2C/delta32 (441 AA form), MEF-2D00, MEF-2D0B, MEF-2DA0, MEF-2DA′0,MEF-2DAB, MEF-2DA′B, Meis-1, Meis-2a, Meis-2b, Meis-2c, Meis-2d,Meis-2e, Meis3, Meox1, Meox1a, Meox2, MHox (K-2), Mi, MIF-1, Miz-1,MM-1, MOP3, MR, Msx-1, Msx-2, MTB-Zf, MTF-1, mtTF1, Mxi1, Myb, Myc, Myc1, Myf-3, Myf-4, Myf-5, Myf-6, MyoD, MZF-1, NC1, NC2, NCX, NELF, NER1,Net, NF III-a, NF III-c, NF III-e, NF-1, NF-1A, NF-1B, NF-1X, NF-4FA,NF-4FB, NF-4FC, NF-A, NF-AB, NFAT-1, NF-AT3, NF-Atc, NF-Atp, NF-Atx,NfbetaA, NF-CLE0a, NF-CLE0b, NFdeltaE3A, NFdeltaE3B, NFdeltaE3C,NFdeltaE4A, NFdeltaE4B, NFdeltaE4C, Nfe, NF-E, NF-E2, NF-E2 p45, NF-E3,NFE-6, NF-Gma, NF-GMb, NF-IL-2A, NF-IL-2B, NF-jun, NF-kappaB,NF-kappaB(-like), NF-kappaB1, NF-kappaB1, precursor, NF-kappaB2,NF-kappaB2 (p49), NF-kappaB2 precursor, NF-kappaE1, NF-kappaE2,NF-kappaE3, NF-MHCIIA, NF-MHCIIB, NF-muE1, NF-muE2, NF-muE3, NF-S, NF-X,NF-X1, NF-X2, NF-X3, NF-Xc, NF-YA, NF-Zc, NF-Zz, NHP-1, NHP-2, NHP3,NHP4, NKX2-5, NKX2B, NKX2C, NKX2G, NKX3A, NKX3A v1, NKX3A v2, NKX3A v3,NKX3A v4, NKX3B, NKX6A, Nmi, N-Myc, N-Oct-2alpha, N-Oct-2beta, N-Oct-3,N-Oct-4, N-Oct-5a, N-Oct-Sb, NP-TCII, NR2E3, NR4A2, Nrf1, Nrf-1, Nrf2,NRF-2beta1, NRF-2gamma1, NRL, NRSF form 1, NRSF form 2, NTF, O2, OCA-B,Oct-1, Oct-2, Oct-2.1, Oct-2B, Oct-2C, Oct-4A, Oct4B, Oct-5, Oct-6,Octa-factor, octamer-binding factor, oct-B2, oct-B3, Otx1, Otx2, OZF,p107, p130, p28 modulator, p300, p38erg, p45, p49erg, -p53, p55, p55erg,p65delta, p67, Pax-1, Pax-2, Pax-3, Pax-3A, Pax-3B, Pax-4, Pax-5, Pax-6,Pax-6/Pd-5a, Pax-7, Pax-8, Pax-8a, Pax-8b, Pax-8c, Pax-8d, Pax-8e,Pax-8f, Pax-9, Pbx-1a, Pbx-1b, Pbx-2, Pbx-3a, Pbx-3b, PC2, PC4, PC5,PEA3, PEBP2alpha, PEBP2beta, Pit-1, PITX1, PITX2, PITX3, PKNOX1, PLZF,PO-B, Pontin52, PPARalpha, PPARbeta, PPARgamma1, PPARgamma2, PPUR, PR,PR A, pRb, PRD1-BF1, PRDI-BFc, Prop-1, PSE1, P-TEFb, PTF, PTFalpha,PTFbeta, PTFdelta, PTFgamma, Pu box binding factor, Pu box bindingfactor (BJA-B), PU.1, PuF, Pur factor, R1, R2, RAR-alpha1, RAR-beta,RAR-beta2, RAR-gamma, RAR-gamma1, RBP60, RBP-Jkappa, Rel, RelA, RelB,RFX, RFX1, RFX2, RFX3, RFX5, RF-Y, RORalpha1, RORalpha2, RORalpha3,RORbeta, RORgamma, Rox, RPF1, RPGalpha, RREB-1, RSRFC4, RSRFC9, RVF,RXR-alpha, RXR-beta, SAP-1a, SAP1b, SF-1, SHOX2a, SHOX2b, SHOXa, SHOXb,SHP, SIII-p110, SIII-p15, SIII-p18, SIM1, Six-1, Six-2, Six-3, Six-4,Six-5, Six-6, SMAD-1, SMAD-2, SMAD-3, SMAD-4, SMAD-5, SOX-11, SOX-12,Sox-4, Sox-5, SOX-9, Sp1, Sp2, Sp3, Sp4, Sph factor, Spi-B, SPIN, SRCAP,SREBP-1a, SREBP-1b, SREBP-1c, SREBP-2, SRE-ZBP, SRF, SRY, SRP1, Staf-50,STAT1alpha, STAT1beta, STAT2, STAT3, STAT4, STATE, T3R, T3R-alpha1,T3R-alpha2, T3R-beta, TAF(I)110, TAF(I)48, TAF(I)63, TAF(II)100,TAF(II)125, TAF(II)135, TAF(II)170, TAF(II)18, TAF(II)20, TAF(II)250,TAF(II)250Delta, TAF(II)28, TAF(II)30, TAF(II)31, TAF(II)55,TAF(II)70-alpha, TAF(II)70-beta, TAF(II)70-gamma, TAF-I, TAF-II, TAF-L,Ta1-1, Ta1-1beta, Ta1-2, TAR factor, TBP, TBX1A, TBX1 B, TBX2, TBX4,TBX5 (long isoform), TBX5 (short isoform), TCF, TCF-1, TCF-1A, TCF-1B,TCF-1C, TCF-1D, TCF-1E, TCF-1F, TCF-1G, TCF-2alpha, TCF-3, TCF-4,TCF-4(K), TCF-4B, TCF-4E, TCFbeta1, TEF-1, TEF-2, te1, TFE3, TFEB,TFIIA, TFIIA-alpha/beta precursor, TFIIA-alpha/beta precursor,TFIIA-gamma, TFIIB, TFIID, TFIIE, TFIIE-alpha, TFIIE-beta, TFIIF,TFIIF-alpha, TFIIF-beta, TFIIH, TFIIH*, TFIIH-CAK, TFIIH-cyclin H,TFIIH-ERCC2/CAK, TFIIH-MAT1, TFIIH-MO15, TFIIH-p34, TFIIH-p44,TFIIH-p62, TFIIH-p80, TFIIH-p90, TFII-I, Tf-LF1, Tf-LF2, TGIF, TGIF2,TGT3, THRA1, TIF2, TLE1, TLX3, TMF, TR2, TR2-11, TR2-9, TR3, TR4, TRAP,TREB-1, TREB-2, TREB-3, TREF1, TREF2, TRF (2), TTF-1, TXRE BP, TxREF,UBF, UBP-1, UEF-1, UEF-2, UEF-3, UEF-4, USF1, USF2, USF2b, Vav, Vax-2,VDR, vHNF-1A, vHNF-1B, vHNF-1C, VITF, WSTF, WT1, WT1I, WT1 I-KTS, WT1I-del2, WT1-KTS, WT1-del2, X2BP, XBP-1, XW-V, XX, YAF2, YB-1, YEBP, YY1,ZEB, ZF1, ZF2, ZFX, ZHX1, ZIC2, ZID, ZNF174, amongst others.

An activated transcription factor is a transcription factor that hasbeen activated by a stimulus resulting in a measurable change in thestate of the transcription factor, for example a post-translationalmodification, such as phosphorylation, methylation, and the like.Activation of a transcription factor can result in a change in theaffinity for a particular DNA sequence or of a particular protein, suchas another transcription factor and/or cofactor.

II. Detailed Description of Several Embodiments a. SyntheticOligonucleotides

Disclosed herein are synthetic oligonucleotides useful in the detectionof DNA binding proteins. The oligonucleotides comprise (a) a sequencethat includes a nucleic acid binding protein binding site, (b) asequence that facilitates the amplification of the primer such as abinding site for a PCR primer and/or an RNA polymerase promoter, and (c)a tag sequence. In some examples, the nucleic acid binding proteinbinding site is 20 nucleotides in length. At least the nucleic acidbinding protein binding site is double stranded and the entire syntheticoligonucleotide can be double stranded. In still further examples, theoligonucleotide comprises SEQ ID NO: 195, SEQ ID NO: 196, and/or SEQ IDNOs: 3-98.

The nucleic acid binding protein binding site may be any binding sitefor a protein that can bind a specific nucleic acid sequence. Suchbinding sites include known binding sites, putative binding sites,mutant or otherwise altered binding sites, or even random sets ofnucleotides that are used to seek out unknown nucleic acid bindingproteins. In addition, the nucleic acid binding site may comprisemethylated or otherwise altered nucleic acids.

A tag sequence is an artificial nucleic acid sequence that does not bindany known nucleic acid binding protein. In some examples, a tag sequenceis a non-naturally occurring nucleic acid sequence that does not bindany known nucleic acid binding protein. One of skill in the art in lightof this disclosure will understand how to make a tag sequence or set oftag sequences that can be incorporated into the probes described herein.Additionally, one of skill in the art in light of this disclosure wouldbe able to determine without undue experimentation whether or not aparticular sequence binds to a nucleic acid binding protein and wouldtherefore not use that sequence as a tag sequence. Examples of tagsequences include SEQ ID NOs: 3-98 herein.

In some examples the oligonucleotide probe is between 85 and 130nucleotides in length, between, 88 and 120 nucleotides, between 90 and110 nucleotides, between 88 and 98 nucleotides in length, between 90 and96 nucleotides in length, between 92 and 94 nucleotides in length,including 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 100,101, 102, 103, 104, 105, 106, 107, 018, 019, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, or 130 nucleotides in length. An exemplary set of the syntheticoligonucleotides described herein includes SEQ ID NOs: 99-194 and SEQ IDNOs: 197-214 herein.

In some examples, oligonucleotides described herein contain one or moremodifications. Modified oligonucleotides include those comprisingmodified backbones or non-natural internucleoside linkages. As definedherein, oligonucleotides having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone.

Examples of modified oligonucleotide backbones include, but are notlimited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkyl-phosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of the nucleoside units are linked3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Representative U.S. patents that teachthe preparation of the above phosphorus-containing linkages include, butare not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is hereinincorporated by reference.

Examples of modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. Representative U.S. patents that teach thepreparation of the above oligonucleosides include, but are not limitedto, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134;5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704;5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of whichis herein incorporated by reference.

Modified oligonucleotides can also contain one or more substituted sugarmoieties. In some examples, the oligonucleotides can comprise one of thefollowing at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Oligonucleotides can also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof modified sugar structures include, but are not limited to, U.S. Pat.Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; and 5,700,920, each of which is herein incorporated byreference in its entirety.

Oligonucleotides can also include base modifications or substitutions.As used herein, “unmodified” or “natural” bases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified bases include other synthetic andnatural bases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Further modified bases have been described (see, for example, U.S. Pat.No. 3,687,808; and Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRCPress, 1993).

Certain of these modified bases are useful for increasing the bindingaffinity. For example, 5-methylcytosine substitutions have been shown toincrease nucleic acid duplex stability by 0.6-1.2° C. RepresentativeU.S. patents that teach the preparation of modified bases include, butare not limited to, U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; 5,681,941; and 5,750,692, each of which is hereinincorporated by reference.

Also disclosed is a synthetic oligonucleotide library that includes aplurality of the synthetic oligonucleotides described herein. Thelibrary can contain at least, 2, 16, 48, 96, 384, 1000, 10,000, or100,000 or more different probes. Each synthetic oligonucleotide in thelibrary can have a different nucleic acid binding protein binding site.Each probe can comprise a different nucleic acid protein binding sitethan all the other probes in the library. Additionally, each syntheticoligonucleotide in the library can comprise a different tag sequencethan all the other tag sequences with each tag sequence in the librarycoupled with a particular nucleic acid protein binding site.

b. Oligonucleotide Synthesis

The synthetic oligonucleotides disclosed herein may be synthesized byany method now known in the art or yet to be disclosed. Oligonucleotidesynthesis may be carried out by the addition of nucleotide residues tothe 5′-terminus of a growing chain. Elements of oligonucleotidesynthesis include: de-blocking (detritylation): A DMT group is removedwith a solution of an acid, such as TCA or Dichloroacetic acid (DCA), inan inert solvent (dichloromethane or toluene) and washed out, resultingin a free 5′ hydroxyl group on the first base. Coupling: A nucleosidephosphoramidite (or a mixture of several phosphoramidites) is activatedby an acidic azole catalyst, tetrazole, 2-ethylthiotetrazole,2-bezylthiotetrazole, 4,5-dicyanoimidazole, or a number of similarcompounds. This mixture is brought in contact with the starting solidsupport (first coupling) or oligonucleotide precursor (followingcouplings) whose 5′-hydroxy group reacts with the activatedphosphoramidite moiety of the incoming nucleoside phosphoramidite toform a phosphite triester linkage. The phosphoramidite coupling may becarried out in anhydrous acetonitrile. Unbound reagents and by-productsmay be removed by washing.

A small percentage of the solid support-bound 5′-OH groups (0.1 to 1%)remain unreacted and should be permanently blocked from further chainelongation to prevent the formation of oligonucleotides with an internalbase deletion commonly referred to as (n−1) shortmers. This is done byacetylation of the unreacted 5′-hydroxy groups using a mixture of aceticanhydride and 1-methylimidazole as a catalyst. Excess reagents areremoved by washing.

The newly formed tricoordinated phosphite triester linkage is of limitedstability under the conditions of oligonucleotide synthesis. Thetreatment of the support-bound material with iodine and water in thepresence of a weak base (pyridine, lutidine, or collidine) oxidizes thephosphite triester into a tetracoordinated phosphate triester, aprotected precursor of the naturally occurring phosphate diesterinternucleosidic linkage. This step can be substituted with asulfurization step to obtain oligonucleotide phosphorothioates. In thelatter case, the sulfurization step is carried out prior to capping.Upon the completion of the chain assembly, the product may be releasedfrom the solid phase to solution, deprotected, and collected. Productsmay be isolated by HPLC to obtain the desired oligonucleotides in highpurity.

c. Oligonucleotide Labeling

The hybridized synthetic oligonucleotides can be detected by detectingone or more labels bonded to the sample nucleic acids. The labels can beincorporated by any of a number of methods. In one example, the label issimultaneously incorporated during nucleic acid amplification. Thus, forexample, polymerase chain reaction (PCR) with labeled primers or labelednucleotides will provide a labeled amplification product. Alternatively,transcription amplification using an RNA polymerase and a labelednucleotide (such as fluorescein-labeled UTP and/or CTP) can be used toincorporate a label into the transcribed nucleic acids.

Alternatively, a label may be added directly to the original nucleicacid sample (such as mRNA, polyA mRNA, cDNA, etc.) or to theamplification product after the amplification is completed. Methods ofattaching labels to nucleic acids are well known to those of skill inthe art and include, for example, nick translation or end-labeling (e.g.with a labeled RNA) by phosphorylation of the nucleic acid andsubsequent attachment (ligation) of a nucleic acid linker joining thesample nucleic acid to a label (e.g., a fluorophore).

Detectable labels suitable for use include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical, chemical, or other detection systems. Useful labels includebiotin for staining with labeled streptavidin conjugate, magnetic beads(for example DYNABEADS™), fluorescent dyes (for example, fluorescein,Texas red, rhodamine, green fluorescent protein, and the like),radiolabels (for example, 3H, 125I, 35S, 14C, or 32P), enzymes (forexample, horseradish peroxidase, alkaline phosphatase and otherscommonly used in an ELISA), and colorimetric labels such as colloidalgold or colored glass or plastic (for example, polystyrene,polypropylene, latex, etc.) beads. Patents teaching the use of suchlabels include U.S. Pat. No. 3,817,837; U.S. Pat. No. 3,850,752; U.S.Pat. No. 3,939,350; U.S. Pat. No. 3,996,345; U.S. Pat. No. 4,277,437;U.S. Pat. No. 4,275,149; and U.S. Pat. No. 4,366,241.

Methods of detecting such labels are also well known. Thus, for example,radiolabels may be detected using photographic film or scintillationcounters, fluorescent markers may be detected using a photodetector todetect emitted light. Enzymatic labels are typically detected byproviding the enzyme with a substrate and detecting the reaction productproduced by the action of the enzyme on the substrate, and fluorescentand colorimetric labels are detected by visualizing the colored label.

The label may be added to the synthetic oligonucleotide prior to, orafter amplification and/or hybridization to the array. Some detectablelabels are directly attached to or incorporated into the syntheticoligonucleotide prior to hybridization. Other labels are joined to thesynthetic oligonucleotide after hybridization to the array. Often, theindirect label is attached to a binding moiety that has been attached tothe target nucleic acid prior to the hybridization. Thus, for example,the target nucleic acid can be biotinylated before the hybridization.After hybridization, an avidin-conjugated fluorophore can bind thebiotin hybrid duplexes comprising the biotin, thereby providing a labelthat is easily detected (see Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P.Tijssen, ed. Elsevier, N.Y., 1993).

d. Kits

Also disclosed is a kit comprising the synthetic oligonucleotide librarythat comprises the synthetic oligonucleotides disclosed herein and anarray that comprises at least 2, 16, 48, 96, 384, 1000, 10,000 or100,000 bound synthetic oligonucleotides with the bound syntheticoligonucleotides bound to a solid substrate. Each bound syntheticoligonucleotide comprises a sequence that is complementary (binds viaWatson-Crick base pairing—A-T, C-G) to the tag sequence on one of thesynthetic oligonucleotides in the library. The bound syntheticoligonucleotides are addressable within the context of the microarraysuch that a known synthetic oligonucleotide can bind to a boundoligonucleotide comprising the complement to its tag sequence in a knownlocation on the array. Detection of the binding of the syntheticoligonucleotide to an addressed position on the array further canidentify the nucleic acid binding protein binding site and, in turnindicate that the nucleic acid binding protein was originally present ina sample. Examples of complements to tag sequences in the boundsynthetic oligonucleotides are the complementary sequences of SEQ IDNOs: 3-98.

e. Methods of Use

Also disclosed is a method of detecting the presence of a DNA bindingprotein in a sample. This method comprises, contacting a library ofsynthetic oligonucleotides described herein with a sample that maycontain a nucleic acid binding protein. The synthetic oligonucleotidesare allowed to bind to the nucleic acid binding protein thereby formingcomplexes. The complexes are purified by electrophoresis and a label isincorporated into the synthetic oligonucleotide. The syntheticoligonucleotide is hybridized to an array comprising bound syntheticoligonucleotides as described above and the presence of the label isdetected on the array. Detection of the label on a specific address onthe array is an indication that a nucleic acid binding protein that canspecifically bind the nucleic acid binding site on the syntheticoligonucleotide that is addressed to the specific address on the arrayvia its tag sequence was originally present in the sample.

The label may be any label including a fluorescent label. Incorporationof the label may be by any method including any method that amplifies ortranscribes the nucleic acid such as by performing PCR amplification orcontacting the complex with an RNA polymerase. The electrophoresis maybe any type of electrophoresis including electrophoresis performed usingthe method and device described in US Patent Application Publicationnumber 2012/0160683 (which is incorporated by reference herein in itsentirety.)

For example, the disclosed oligonucleotides may be used withelectrophoresis which utilizes a cassette comprising a proximal end anda distal end opposite the proximal end, a front face and a back facesubstantially parallel to the front face, and at least one chamberdefined between the front face and the back face, the chamber having anupper portion and a lower portion, wherein the upper portion includes anupper opening at or near the proximal end of the cassette and the lowerportion includes a lower opening at or near the distal end of thecassette, and wherein the front face comprises at least one windowopening into the upper portion of the chamber. In some embodiments, thecassette can further comprise a semi-permeable membrane covering atleast a portion of the at least one window opening. The semi-permeablemembrane can be removably secured to the front face of the cassette. Thesemi-permeable membrane can be configured to allow passage of a buffersolution surrounding at least a portion of the cassette, and to at leastpartially block passage of a biomolecule contained within the upperportion of the at least one chamber. In some embodiments, asemi-permeable membrane can substantially block passage of a biomoleculeof interest, thus substantially preventing it from exiting the chamberthrough the window opening. Some embodiments may utilize a cassettecomprising acrylic. Some embodiments of a cassette comprise a unitarybody. Cassettes can be integrated with one or more other components toform an electrophoresis device.

One embodiment of an electrophoresis device which can be utilized in thedisclosed methods with the disclosed oligonucleotides comprises acassette, wherein the cassette comprises a proximal end and a distal endopposite the proximal end, a front face and a back face substantiallyparallel to the front face, and at least one chamber defined between thefront face and the back face, the chamber having an upper portion and alower portion, wherein the upper portion includes an upper opening at ornear the proximal end of the cassette and the lower portion includes alower opening at or near the distal end of the cassette, and wherein thefront face comprises at least one window opening into the upper portionof the chamber. The electrophoresis device can further comprise a firstbuffer solution in fluid contact with the lower opening of the lowerportion, a second buffer solution in fluid contact with the windowopening of the upper portion, and at least one electrode electricallycoupled to each of the first and second buffer solutions. Fluid contactcan include both direct and indirect fluid contact. For example, fluidcontact can include fluid contact through a membrane, such as asemi-permeable membrane.

In some embodiments of an electrophoresis device, the cassette furthercomprises a semi-permeable membrane covering at least a portion of thewindow opening of the upper portion, such as a membrane comprisingcellulose or cellophane. In one particular embodiment, theelectrophoresis device, comprises a first buffer container; and acassette capable of being coupled to the first buffer container to forma first sidewall of the first buffer container, wherein the cassettecomprises a proximal end and a distal end opposite the proximal end, afront face and a back face and at least one chamber defined between thefront face and the back face, the chamber having an upper portion and alower portion, wherein the upper portion includes a proximal opening ator near the proximal end of the cassette and the lower portion includesa distal opening at or near the distal end of the cassette wherein theproximal opening and distal opening are aligned to form a straightpassageway there through, and wherein the front face comprises at leastone window opening into the upper portion of the chamber.

The disclosed oligonucleotides can be used with reversible currentelectrophoresis, such that when current is run in a first direction,free molecules elute into the buffer at the distal end of the device.Then the cassette body can be placed into a new bath, the currentreversed, and the biomolecules of interest can then move in the oppositedirection, out of the gel, and back into the buffer solution at theproximal end of the cassette body, where they can then be easilycollected from the device.

For example, one method of separating a biomolecule of interest from asample comprises providing a sample, wherein the sample contains abiomolecule of interest and free probes, loading the sample onto a gelin a lower portion of a cassette body, electrophoresing the sample byapplying a current for a time period sufficient for substantially all ofthe free probes (such as one or more of the disclosed oligonucleotides)to elute out of a distal end of the lower portion, into a first buffersolution in a first buffer container, leaving the biomolecule ofinterest within the gel, removing the first buffer solution, providing anew buffer solution in fluid contact with the lower portion of thecassette, reversing the current with respect to the gel, andelectrophoresing the sample for a time period sufficient forsubstantially all of the biomolecule of interest to elute out of aproximal end of the lower portion, into an upper portion of the cassettebody.

Disclosed methods can further comprise collecting biomolecule ofinterest. Collecting the biomolecule of interest can comprisewithdrawing a volume of buffer solution containing the biomolecule ofinterest from the upper portion of the cassette body. Collecting thebiomolecule of interest can comprise securing a semi-permeable membraneover at least part of the upper portion, so as to prevent substantiallyall of the biomolecule of interest from passing through thesemi-permeable membrane into a second buffer solution in fluid contactwith the upper portion of the cassette.

In some methods, reversing the current with respect to the gel comprisesswitching the polarity of a first and second electrode in electricalcontact with the first and second buffer solutions, respectively. Insome methods, reversing the current with respect to the gel compriseschanging the orientation of the gel with respect to a first and secondelectrode in electrical contact with the first and second buffersolutions, respectively.

Removing the first buffer solution can comprise draining the firstbuffer solution from a buffer container. Providing a new buffer solutionin fluid contact with the lower portion of the cassette can compriserefilling the buffer container with the new buffer solution. In othermethods, removing the first buffer solution comprises removing thecassette from a first buffer container containing the first buffersolution, and providing a new buffer solution in fluid contact with thelower portion of the cassette comprises placing the cassette in a secondbuffer container containing the new buffer solution.

EXAMPLES

The following examples are illustrative of disclosed methods. In lightof this disclosure, those of skill in the art will recognize thatvariations of these examples and other examples of the disclosed methodwould be possible without undue experimentation.

Example 1 Sample Preparation

Nuclear protein extracts were prepared according to the QIAGEN® protocolusing a Qproteome Nuclear Protein Kit (Catalog number 37582, QIAGEN®,Valencia, Calif.) from 20 to 25 mg tissue or 10 million cells asappropriate. The nuclear protein extracts were aliquotted in 5 μgvolumes and stored at −80° C. prior to the reaction.

A blocking reaction was then performed. The blocking reaction includedPoly(dI-dC) a double stranded alternating copolymer that is commonlyused as a blocking agent in EMSA assays at a concentration of 1 μg/mlper 1 μg/ml of nuclear protein as well as a mixture of polynucleotidesconsisting of equal proportions of SEQ ID NOs 3-98. This mixture wasadded at 0.4 μg/ml per 1 μg/ml of nuclear protein. Exact concentrationsand volumes used are as follows:

Nuclear protein [1.0 μg/μl] 5 μl Poly(IC) [0.5 μg/μl] 2 μl Probe mix[200 ng/μl] 2 μl Add water to 15 μl The blocking reaction lasted for 15 minutes at room temperature.

The blocked reaction mixture was then mixed with a set ofoligonucleotide probes consisting of polynucleotides of SEQ ID NOs:197-214 in the following mixture:

10x buffer 3.0 μl 50% glycerol 1.5 μl 25 mM DTT/2.5% Tween ®-20 3.0 μlProbe set (38 ng/μl) 1.5 μl Water 6.0 μl

This mixture was incubated for 25 minutes at room temperature.

Probes were then run on a gel electrophoresis system configured toseparate and collect complexes comprising transcription factor and boundprobe from free probe. This system is described in US Patent Application2012/0160683, which is incorporated by reference herein.

A 6% acrylamide gel was prepared as follows:

30% acrylamide 4 ml 7.5x TBE 1.33 ml Water 14.6 ml 10% APS 100 μl TEMED10 μlThe running buffer was a 0.5×Tris/borate/EDTA (TBE) buffer.

The gel was run for 135 minutes, a time previously determined to besufficient to clear all unbound probe out of the distal end of the gel.The gel cassette was rinsed, a semipermeable membrane installed in thecollection chamber as described in the referenced patent application,and fresh TBE buffer added. At this point, the electrode polarity isreversed and the gel run with electrode polarity reversed for 210minutes. After the 210 minute electrophoresis, the transcription factorprotein/probe complexes are collected in about a 1.5 ml volume. The 1.5ml volume is concentrated to 50 μl using an Amicon 10 k filter unit andthe samples then purified using a QIAamp nucleotide removal kit. Thevolume of the eluate with the purified probes is 50 μl.

Amplification and labeling was performed using a T7 High Yield RNA®Synthesis Kit (New England Biolabs). Biotinylated UTP is added to theribonucleotide solution from the kit to a level where the biotinylatedUTP makes up 33% of the UTP in the solution. The amplification andlabeling mixture is as follows:

10x buffer (from kit)   2 μl Ribonucleotide mix 7.5 μl (100 mM each ATP,CTP, GTP, 50 mM UTP and 25 mM biotinylated UTP) Purified probes fromelectrophoresis 6.0 μl T7 RNA polymerase (from kit) 2.0 μl Water 2.5 μlAmplification/labeling was performed for 2 hours at 37° C., followingwhich the samples are placed on ice.

Labeled RNA was purified using a QIAGEN® RNeasy Mini-elute Cleanup kit.

Microarray slides were prepared by Microarrays Inc (Huntsville, Ala.).Four subarrays of 25 nucleotide-long tag sequences were printed perslide. Complements of ninety-six tag sequences (SEQ ID NOs: 3-98) wereused for each subarray. Each tag sequence was printed 4 times—once ineach subarray.

Labeled RNA was mixed with hybridization buffers A and B (Hyb & FragBuffers, CodeLink®, Applied Microarrays) and hybridized to eachmicroarray under the following conditions:

Samples were heated at 68° C. for 3 minutes and then placed on ice for 2minutes. Then 55 μl of sample was injected into a Tecan 400 prohybridization station at 37° C. for 18 hours. Samples were washed with0.75 TNT fast wash at room temperature, 2 times, then incubated in 0.75TNT at 37° C. for 1 hour.

A Cy5-Streptavidin working solution was prepared by mixing 1 μCy5 with499 μl TNB buffer. A 55 μl volume of Cy5-Streptavidin working solutionwas injected into the sample through a positive pressure pipette andincubate at room temperature for 30 min. Slides were then washed with1×TNT wash 4 times at 5 minutes per wash. Slides were then washed with0.1×SSC/0.05% Tween 20 at room temperature for 30 seconds. Slides werethen dried by nitrogen flow.

A GenePix 4000B Microarray scanner was used to scan the slide using asingle wavelength scan at 635 nm. The PMT gain ranged from 450-650.

Normalization of signal was performed as follows:Average signal of the subarray=total signal of the subarray/(96×4)Average signal of the probe=total signal of the probe/4Relative strength of a probe=Average signal of the probe/Average signalof the subarray

Example 2 Quantifiable Detection of NF-κB in Liver Cells

Liver nuclear extract was used as the control (group 1), 10 ng (group2), 50 ng (group 3) and 100 ng (group 4) of purified NF-Kb protein wereadded to same amount of liver nuclear extract and then bind to a groupof mixed probes, which included NF-Kb probes, ER-alpha probes, SP-1probes, mutated NF-Kb probes, mutated ER-alpha probes, and mutated SP-1probes. The probes that bind to nuclear protein in the samples wereseparated with our gel device and then labeled and hybridized toindividual subarray of microarray slide. The relative strength of oneprobes were calculated as:Relative strength=average signal of one probe/average signal of thesubarray

Results are shown in FIG. 1. Relative strength is calculated on the Yaxis. The relative strength of three NF-Kb probes (SEQ ID NO: 197, SEQID NO: 198, and SEQ ID NO: 99) increased in relation in nuclear extractswith added NF-κb. A mutant probe for NF-κB (SEQ ID NO: 202) did notincrease and did decrease in relative strength with the addition ofNF-κB.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A synthetic oligonucleotide comprising: a first sequencecomprising a nucleic acid binding protein binding site; a secondsequence comprising a binding site for a first PCR primer or an RNApolymerase promoter; and a third sequence comprising a tag sequence,wherein the tag sequence binds no nucleic acid binding protein andwherein the tag sequence is at least 15 nucleotides long; wherein atleast the first sequence is double stranded and wherein the total lengthof the oligonucleotide is between 88 and 98 nucleotides.
 2. Thesynthetic oligonucleotide of claim 1, wherein the second sequence is SEQID NO: 195 or SEQ ID NO:
 196. 3. The synthetic oligonucleotide of claim2, further comprising a fourth sequence, the fourth sequence comprisinga binding site for a second PCR primer.
 4. The synthetic oligonucleotideof claim 1, wherein the entire oligonucleotide is double-stranded. 5.The synthetic oligonucleotide of claim 1, comprising one or more copiesof a single nucleic acid binding protein binding site.
 6. The syntheticoligonucleotide of claim 1, wherein the oligonucleotide is between 90and 98 nucleotides in length.
 7. The synthetic oligonucleotide of claim1, comprising SEQ ID NO: 1 and/or SEQ ID NO:
 2. 8. The syntheticoligonucleotide of claim 1, wherein the tag sequence is 25 nucleotidesin length.
 9. The synthetic oligonucleotide of claim 8, comprising anyof SEQ ID NOs: 3-98.
 10. A method of detecting the presence of a nucleicacid binding protein in a sample, the method comprising: contacting asynthetic oligonucleotide of claim 1 with a sample, the samplecomprising a nucleic acid binding protein; subjecting the sample toconditions that allow binding of the DNA binding protein to thesynthetic oligonucleotide to form a nucleic acid bindingprotein/oligonucleotide complex; purifying the nucleic acid bindingprotein/oligonucleotide complex by electrophoresis; denaturing thenucleic acid binding protein/oligonucleotide complex; incorporating alabel into the synthetic oligonucleotide; hybridizing the syntheticoligonucleotide to an array, wherein the array comprises a boundoligonucleotide, the bound oligonucleotide comprising the complement ofthe tag sequence and detecting the presence of the label; wherein thehybridizing the synthetic oligonucleotide to the array is performedafter the purifying the nucleic acid binding protein/oligonucleotidecomplex by electrophoresis.
 11. The method of claim 10, wherein thelabel is a fluorescent label.
 12. The method of claim 10, whereinincorporating the label into the synthetic oligonucleotide comprisesperforming PCR amplification with primers complementary to a universalPCR forward primer sequence and a universal PCR reverse primer sequence.13. The method of claim 10, wherein incorporating the label into thesynthetic oligonucleotide comprises contacting the syntheticoligonucleotide with an RNA polymerase.
 14. The method of claim 10,wherein the electrophoresis is performed using an electrophoresis devicecomprising: a cassette, wherein the cassette comprises a proximal endand a distal end opposite the proximal end, a front face and a back faceand at least one chamber defined between the front face and the backface, the chamber having an upper portion and a lower portion, whereinthe upper portion includes an upper opening at or near the proximal endof the cassette and the lower portion includes a lower opening at ornear the distal end of the cassette, and wherein the front facecomprises at least one window opening into the upper portion of thechamber; a first buffer solution in fluid contact with the lower openingof the lower portion; a second buffer solution in fluid contact with thewindow opening of the upper portion; and at least one electrodeelectrically coupled to each of the first and second buffer solutions.15. A synthetic oligonucleotide library comprising: a first syntheticoligonucleotide of claim 1, the first synthetic oligonucleotidecomprising a first nucleic acid binding protein binding site and a firsttag sequence; and a second synthetic oligonucleotide of claim 1, thesecond synthetic oligonucleotide comprising a second nucleic acidbinding protein binding site and a second tag sequence; wherein thefirst nucleic acid binding protein binding site and the second nucleicacid binding protein binding site are different sequences.
 16. Thesynthetic oligonucleotide library of claim 15, wherein the first tagsequence is different from the second tag sequence.
 17. The syntheticoligonucleotide library of claim 15, comprising two or more of theprobes of SEQ ID NOs: 99-195 or SEQ ID NOs: 197-214.
 18. A kitcomprising: the synthetic oligonucleotide library of claim 15; and anarray, the array comprising a third synthetic oligonucleotide comprisingthe complement of the first tag sequence and a fourth syntheticoligonucleotide comprising the complement of the second tag sequence;wherein the third synthetic oligonucleotide and the fourth syntheticoligonucleotide are each addressable on the microarray.
 19. The kit ofclaim 18, wherein the third synthetic oligonucleotide and the fourthsynthetic oligonucleotide comprise the complements of any of SEQ ID NOs:3-98.
 20. The synthetic oligonucleotide probe of claim 1, wherein asignal detected in a microarray is increased after the syntheticoligonucleotide probe is contacted with a nucleic acid binding proteinthat can bind the nucleic acid binding protein binding site.